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To overcome poor treatment response of pediatric high-risk acute lymphoblastic leukemia (ALL), novel treatment strategies are required to reactivate programmed cell death in this malignancy. Therefore, we take advantage of using small-molecule antagonists of Inhibitor of apoptosis (IAP) proteins, so called Smac mimetics such as BV6, which are described to overcome apoptosis resistance and thereby sensitize tumor cells for several apoptotic stimuli. To address the question whether redox alterations can sensitize leukemic cells for Smac mimetic-mediated cell death, we interfered with the cellular redox status in different ALL cell lines. Here, we show for the first time that redox alterations, mediated by the glutathione depleting agent Buthioninesulfoximine (BSO), prime ALL cells for BV6-induced apoptosis. Besides ALL cell lines, BV6/BSO cotreatment similarly synergizes in cell death induction in patient-derived primary leukemic samples. In contrast, the combination treatment does not exert any cytotoxicity against peripheral blood lymphocytes (PBLs) or mesenchymal stroma cells (MSCs) from healthy donors, suggesting some tumor selectivity of this treatment. We also identify the underlying molecular mechanism of the novel synergistic drug interaction of BSO and BV6. We demonstrate that both agents act in concert to increase reactive oxygen species (ROS) production, lipid peroxidation and finally apoptotic cell death. Enhanced ROS levels in the combination treatment account for cell death induction, since several ROS scavengers, like NAC, MnTBAP and Trolox attenuate BSO/BV6-induced apoptosis. BSO/BV6-induced ROS can be mainly classified as lipid peroxides, since the vitamin E derivate α-Tocopherol as well as Glutathione peroxidase 4 (GPX4), which both specifically reduce lipid-membrane peroxides, prevent lipid peroxidation, caspase activation and cell death induction. Vice versa, GPX4 knockdown and pharmacological inhibition of GPX4 by RSL3 or Erastin enhance BV6-induced cell death. Importantly, cell death induction critically depends on the formation of a complex consisting of RIP1/FADD/Caspase-8, since all complex components are required for ROS production, lipid peroxidation and cell death induction. Taken together, we demonstrate that BSO and BV6 cooperate to induce ROS production and lipid peroxidation which are eventually required for caspase activation and cell death execution. Collectively, findings of this study indicate that BV6-induced apoptosis is mediated via redox alterations offering promising new treatment strategy to overcome apoptosis resistance in ALL.
Recently, two of the most common types of bone cancers in children and young adults have been proven to exhibit vulnerability to poly(ADP)-ribose polymerase, (PARP) inhibitors (e.g. olaparib, talazoparib). Ewing’s sarcoma (ES) are reported to harbor a fusion gene EWS-FLI1 (85%), inducing tumorigenesis. Additional, as the fusion gene acts as aberrant transcription factor, it similarly induces elevated PARP expression levels sensitizing ES to PARP inhibition. Second, by an exome sequencing approach in a set of primary osteosarcomas (OS) we identified mutation signatures being reminiscent of BRCA deficiency. Therefore, the sensitivity of a panel of OS cell lines to either talazoparib single treatment or in combination with several chemotherapeutic drugs was investigated.
To screen ES tumor cell lines against PARP inhibitors we applied four different PARP inhibitors (talazoparib, olaparib, niraparib and veliparib) that are frequently being used for clinical studies. We combined those PARP inhibitors with a set of chemotherapeutics (temozolomide (TMZ), SN-38, etoposide, ifosfamide, doxorubicin, vincristine and actinomycin D) that are part of the first-line therapy of ES patients. Here, we demonstrate how PARP inhibitors synergize with TMZ or SN-38 to induce apoptosis, whereas the combination of PARP inhibitors with the other drugs are not favorable. By investigation of key checkpoints in the molecular mechanisms of cell death, the pivotal role of the mitochondrial pathway of apoptosis mediating the synergy between olaparib and TMZ was revealed.
Employing talazoparib monotherapy in combination with or without several chemotherapeutic drugs (TMZ, SN-38, cisplatin, doxorubicin, methotrexate and etoposide/carboplatin), the correlation between homologous recombination (HR) repair deficiency (BRCAness) and the response to talazoparib as prototypical PARP inhibitor was validated in different OS cell lines. By calculation of combination indices (CI) and fraction affected (Fa) values, we identified TMZ as the most potent chemotherapeutic drug in combination with talazoparib inducing the mitochondrial apoptotic pathway in OS.
In our studies of two independent tumor entities with contrary genetic background we identified the combination of PARP inhibitor and TMZ as being most effective. Our studies point out that after TMZ induced DNA methylation and concomitant PARP trapping, DNA damage-imposed checkpoint kinase activation consequently induces G2-cell cycle arrest. Subsequent, PARP inhibitor/TMZ causes MCL-1 degradation, followed by activation of BAK and BAX, succeeding in loss of mitochondrial outer membrane potential (LMMP) and activation of downstream effector-caspases in mitochondrial apoptosis. Our findings emphasize the importance of PARP inhibition in order to chemosensitize ES, which express high PARP levels, or OS that bear features of BRCAness.
Acute lymphoblastic leukemia (ALL), a neoplastic disorder of blood cells of the lymphoid lineage, is the most frequent childhood cancer. In spite of increasing survival rates, the outcome for adults, infants or relapsed patients is still less favorable, highlighting the need for novel treatment options. Reactive oxygen species (ROS) are important signaling molecules that are involved in a variety of cellular pathways. As high ROS levels lead to oxidative stress and irreversible oxidation of cellular macromolecules, the production and elimination of ROS is tightly controlled. Therefore, cells express several antioxidant molecules and enzymes, including glutathione, catalase and the thioredoxin (Trx) system, to balance ROS levels. As cancer cells were found to have increased ROS levels that could contribute to tumor progression and metastasis, they rely strongly on these antioxidant systems to prevent oxidative damage, making cancer cells especially vulnerable to ROS-inducing treatments. ROS and oxidative stress have been shown to induce programmed cell death via different pathways, however the exact mechanisms that couples oxidative signaling and cell death is not completely understood.
As a disturbance of the cellular redox homeostasis was reported during leukemia development and progression, we wanted to determine the potential of Trx inhibitors for ALL therapy. Additionally, we aimed to further understand the role of ROS and subsequent protein oxidation in the induction and execution of programmed cell death.
First, we demonstrated that the Trx1 inhibitor PX-12 induced cell death in three ALL cell lines. Further analysis of the events leading to PX-12-induced cell death in FADD-deficient (FD) Jurkat cells revealed an increase in ROS levels and oxidation-mediated dimer formation of peroxiredoxin 3 (PRDX3). Interestingly cell death was inhibited by the thiol-containing antioxidant N-acetylcysteine (NAC), but not by non-thiol-containing ROS scavengers. PX-12 treatment further induced cleavage of caspase-9 and -3 and activation of the pro-apoptotic BCL-2 protein BAK, leading us to the conclusion that mitochondria-dependent apoptosis was induced. Interestingly, we could demonstrate an important role for the BH3-only protein NOXA in the mediation of PX-12-induced apoptosis as knock-down of NOXA prevented cell death induction and BAK activation. Our findings give novel insights into the mechanism of PX-12-induced cell death in ALL cell lines and underscores the potential of PX-12 for the treatment of ALL.
To further understand the processes leading to cell death upon inhibition of the Trx system, we analyzed global protein oxidation in Jurkat FD cells upon treatment with the Trx reductase inhibitor Auranofin. In line with previous results, Auranofin induced intrinsic apoptosis that was dependent on BAK and accompanied by increased ROS levels. Using a BIAM Switch Assay followed by mass spectrometry, we demonstrated that Auranofin treatment induced oxidation of over 200 proteins. We identified several proteins whose oxidation upon Auranofin treatment was expected, like Trx1, Trx2 and several peroxiredoxins. Additionally, we verified oxidation of APAF1-interacting protein (APIP) and protein arginine N-methyltransferase (PRMT1) that are both implicated in the regulation of apoptosis. With this analysis we were able to demonstrate that Auranofin treatment leads to changes in global protein oxidation. Whether oxidation of the determined proteins changes their functionality and contributes to apoptosis induction remains to be elucidated.
As we identified BAK as an important player in PX-12- and Auranofin-induced cell death in the previous parts of this study, we wanted to further understand its involvement in ROS-mediated cell death. First analyses in wild-type (WT) and BAK-/- murine embryonic fibroblasts (MEFs) revealed that BAK was essential for Auranofin-induced cell death and that this cell death was caspase-independent in MEFs. Interestingly, BAK oxidation was induced upon treatment with Auranofin, but not upon stimulation with the apoptosis-inducing compound Etoposide. Expression of mutated BAK, with either one or both oxidation-sensitive cysteines mutated to oxidation-insensitive serines, revealed that mutating already one cysteine protected cells from Auranofin , but not Etoposide-induced cell death. Of note, mutation of the BAK BH3 domain rescued MEFs from both, Auranofin- and Etoposide-mediated cell death. The presence of cysteine residues also altered BAK interactions as observed by a mass spectrometric analysis of Auranofin-treated MEFs expressing either WT or cysteine-less BAK. We identified interactions of WT BAK with proteins involved in mitochondrial fission and vesicle transport upon Auranofin treatment. Of note, interaction with proteins involved in apoptosis, like BAX or BCL-XL, was not changed between WT and cysteine-less BAK. Our results demonstrate a critical role for BAK oxidation in Auranofin-induced cell death. Furthermore, we identified novel oxidation-dependent BAK interaction partners.
To conclude, this study highlights the potential of ROS-inducing treatments for ALL therapy and provides novel insights into the redox regulation of programmed cell death.
Inhibitoren der Apoptose (IAP, inhibitor of apoptosis) Proteine spielen eine wichtige Rolle in Bezug auf Zelltodregulation und es ist anzunehmen, dass eine Dysregulation dieser Proteine zu einer Tumorentwicklung und Tumorprogression beiträgt. Erhöhte Expressionslevel von IAP Proteinen verhindern die Aktivierbarkeit des Zelltodprogrammes von Tumorzellen und eine Reihe von Studien konnte bereits erhöhte IAP Level in Tumorzelllinien sowie in primären Tumorproben nachweisen. Des Weiteren korrelieren erhöhte Expressionslevel von IAPs in Tumoren mit Behandlungsresistenzen und schlechten Prognosen für die Patienten.
Das diffuse großzellige B-Zell Lymphom (DLBCL, diffuse large B-cell lymphoma) zählt zu den häufigsten Subtypen der Non-Hodgkin Lymphome (NHL) mit 40 % aller neu diagnostizierten NHL Fälle. DLBCL ist eine sehr heterogene Erkrankung die in drei verschiedene Gruppen klassifiziert wurde: aktivierter B-Zell Typ (ABC, activated B-cell), Keimzentrum B-Zell Typ (GCB, germinal center B-Cell) und Mediastinaler großzelliger B-Zell Typ (PMBL, primary mediastinal B-cell lymphoma). Erhöhte Expressionslevel von zellulärem IAP1 (cIAP, cellular IAP) und cIAP2 wurden ebenfalls in primären Tumorproben von DLBCL Patienten nachgewiesen. Smac mimetics wurden entwickelt, um IAPs zu antagonisieren und stellen damit eine Behandlungsstrategie für DLBCL Patienten dar, denn ca. 40 % aller DLBCL Patienten entwickeln ein Rezidiv oder erreichen gar keine Remission unter Standardtherapie. Jedoch ist der Effekt von Smac mimetics in einer Einzelbehandlung limitiert, weswegen Kombinationstherapien mit Smac mimetics eine vielversprechende Strategie für ihren klinischen Einsatz darstellen. Aus diesem Grund haben wir in dieser Arbeit den Effekt von Smac mimetic in Kombination mit Proteasom-Inhibitoren analysiert und einen speziellen Fokus auf den molekularen Mechanismus des ausgelösten Zelltodsignalweges gelegt.
Die Kombination verschiedener Konzentrationen des Smac mimetics BV6 mit dem Proteasom-Inhibitor carfilzomib (CFZ) löst in allen drei getesteten DLBCL Subtypen (ABC, GCB und PMBL) Zelltod aus. Die Kalkulation des Kombinationsindexes (CI, combination index) sowie des Bliss Scores, zwei quantitative Parameter zur Bestimmung eines Synergismus, zeigen, dass fast alle getesteten Kombinationen einen Synergismus aufweisen. Dies verdeutlicht, dass eine Co-Behandlung von BV6 und CFZ eine wirksame Kombination ist um Zelltod in DLBCL Zelllinien auszulösen. Außerdem zeigt eine Kombination von BV6 mit anderen Proteasom-Inhibitoren wie ixazomib (IXA) oder oprozomib (OPR), ebenfalls eine synergistische Reduktion der Zellviabilität. Diese Ergebnisse deuten darauf hin, dass der detektierte Effekt nicht auf eine Substanz limitiert ist, sondern, dass ein genereller Effekt von Smac mimetic und Proteasom-Inhibitoren vorliegt, um Zellviabilität in DLBCL zu reduzieren. BV6 und CFZ induzieren einen apoptotischen Zelltod, da sie die Spaltung und Aktivierung von Initiator- und Effektorcaspasen (Caspasen-3, -7, -8 und -9) initiieren und sich der induzierte Zelltod mit Hilfe des Caspasen-Inhibitors zVAD.fmk verhindern lässt. Die Behandlung mit BV6 und CFZ führt zu einer Akkumulation von NIK, ein Protein welches zur Aktivierung des non-kanonischen NF-kB Signalweges benötigt wird. Weitere Untersuchungen zeigen jedoch, dass NIK nicht an der Zelltodinduktion beteiligt ist, da eine siRNA-basierte Herunterregulierung des NIK Proteins keinen Einfluss auf die Zelltodinduktion nimmt. Ebenfalls ist der Zelltod unabhängig von dem TNFa Signalweg, da weder eine Behandlung mit dem TNFa Inhibitor Enbrel den Zelltod verringern kann noch eine zusätzliche Gabe von TNFa den Zelltod erhöht. Weitere mechanistische Studien zeigen eine kritische Rolle der mitochondrialen Apoptose für den BV6/CFZ-vermittelten Zelltod. Unter Behandlung mit BV6/CFZ wurde eine Aktivierung von BAX und BAK nachgewiesen, welche beide mit verantwortlich für die Porenbildung in der mitochondrialen Membran sind. Eine Herunterregulation dieser beiden Proteine mittels siRNA reduziert signifikant den durch BV6/CFZ-induzierten Zelltod auf ein Minimum. Gleichzeitig löst eine Co-Behandlung mit BV6/CFZ einen Verlust des mitochondrialen Membranpotentials (LOMMP, loss of mitochondrial membrane potential) aus. In Übereinstimmung mit den vorherigen Experimenten, zeigen wir eine Akkumulation von mitochondrialen reaktiven Sauerstoffspezies (ROS; reactive oxygen species), sowie einen generellen Anstieg des allgemeinen ROS Levels. Eine Behandlung mit BV6/CFZ zeigt eine deutliche Akkumulation des pro-apoptotischen Proteins NOXA. Um dessen funktionelle Relevanz zu überprüfen, wurde die Proteinmenge von NOXA mittels siRNA stark reduziert. Eine Behandlung mit der Kombination aus BV6 und CFZ zeigt daraufhin eine signifikant reduzierte Zelltodinduktion, was die funktionelle Relevanz von NOXA für den BV6/CFZ-vermittelten Zelltod unterstreicht. Immunopräzipitationsstudien zeigen, dass in RIVA und U2932 Zellen NOXA konstitutiv an seinen anti-apoptotischen Bindungspartner MCL-1 gebunden ist, was die Zellen bereits darauf vorbereitet Apoptose zu durchlaufen. Dieses sogenannte „primen“ für Apoptose wird durch die Behandlung mit BV6 und CFZ weiter verstärkt, da es die Bindung zwischen NOXA und MCL-1 weiter erhöht. Dadurch wird die Balance zwischen pro- und anti-apoptotischen Proteinen zu Gunsten der pro-apoptotischen Proteine verschoben und die Induktion von Apoptose begünstigt.
Insgesamt zeigen die Ergebnisse, dass DLBCL Zelllinien sensitiv auf eine Behandlung mit Smac mimetic und Proteasom-Inhibitor reagieren und damit eine mögliche neue Behandlungsstrategie für diese heterogene Tumorerkrankung darstellt.
Reactive oxygen species (ROS) are involved in various signalling mechanisms. Redox homeostasis is important in cancer cells, since they are dependent on upregulated antioxidant defence pathways to cope with elevated ROS levels. Therefore, targeting the antioxidant defence system and/ or increasing ROS to a lethal level may be a feasible strategy to counteract cancer cell progression.
Acute lymphoblastic leukaemia (ALL) is the most frequent malignant childhood cancer, displaying on one side resistance to cell death induction and on the other side elevated ROS levels. Therefore, inducing ferroptosis, a ROS- and iron-dependent cell death pathway might be useful to trigger cell death in ALL as a novel treatment strategy. In the first study of this thesis we observed that RSL3, a glutathione (GSH) peroxidase 4 (GPX4) inhibitor, triggered ROS accumulation and lipid peroxidation which contributed to ferroptotic cell death. These observations were based on suppression of RSL3 stimulated cell death using different ferroptosis inhibitors like Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), as well as iron chelator Deferoxamine (DFO) and the vitamin E derivate α-Tocopherol (α-Toc). RSL3-triggered ROS and lipid peroxide production were also inhibited through Fer-1 and α-Toc. Furthermore, lipoxygenases (LOX) were activated upon RSL3 stimulation and contributed to ferroptotic cell death in ALL as well. Selective inhibition of LOX with the 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) abolished RSL3-induced ROS production, lipid peroxidation and cell death. In addition, RSL3 induced lipid peroxide-dependent ferroptotic cell death in FAS-associated Death Domain (FADD)-deficient, death receptor-induced apoptosis resistant cells, demonstrating that ferroptosis might circumvent apoptosis resistance.
The second part of the study revealed that RSL3 and Erastin (Era), a GSH-depleting agent, inhibiting the cystine/glutamate antiporter system xc- and ferroptosis inducer, cooperated with the Smac mimetic BV6 to trigger cell death in ALL cells. RSL3/BV6 and Era/BV6 combination-induced cell death was dependent on ROS accumulation, but independent of caspases and key modulators of necroptosis. RSL3/BV6-treated ALL cells exhibited classical features of ferroptotic cell death with iron-dependency, ROS accumulation and lipid peroxidation which was diminished through either pharmacological inhibition (Fer-1, DFO, α-Toc) or genetic inhibition by overexpressing GPX4. Interestingly, Era/BV6-induced cell death in ALL cells was independent of iron but dependent on ROS accumulation, since α-Toc rescued from Era/BV6-triggered ROS production, lipid peroxidation and cell death. Moreover, inhibition of lipid peroxide formation through the addition of Fer-1 or by overexpressing GPX4 failed to rescue from Era/BV6-triggered cell death, even if Era/BV6-stimulated lipid peroxidation was diminished. Likewise, Fer-1 protected from RSL3/BV6-, but not from Era/BV6-generated ROS production, leading to the assumption that other ROS besides lipid-based ROS contributed to cell death in Era/BV6-treated cells. In summary, while RSL3/BV6 induced ferroptosis in ALL, Era/BV6 stimulated a ROS dependent cell death, which was neither dependent on iron nor caspases or receptor-interacting protein (RIP) kinase 1 nor 3. Additionally, using Erastin alone did not trigger ferroptotic cell death in ALL. Finally, with these two studies we tried to unravel the molecular pathway of ferroptosis by using RSL3 and Erastin as well described ferroptosis stimulators. Here, we demonstrate the possibility of a novel treatment strategy to reactivate programmed cell death by impeding redox homeostasis in ALL.
Since ALL failed to induce ferroptosis upon Erastin treatment, we investigated in the third part of this thesis a new model system to induce ferroptosis upon Erastin and RSL3 exposure. Previous studies revealed that rhabdomyosarcoma (RMS) cells might be susceptible to oxidative stress-induced compounds. To this end, we used Erastin as a prototypic ferroptosis stimulus and GSH-depleting agent and demonstrated that GSH depletion, ROS and lipid ROS accumulation contributed to cell death. Additionally, Fer-1, Lip-1, DFO, lipophilic vitamin E derivate α-Toc and GSH, a cofactor of GPX4, protected from Erastin stimulated ROS accumulation, lipid peroxidation and cell death. Also, the use of a broad spectrum protein kinase C (PKC) inhibitor Bisindolylmaleimide I (Bim1), a PKCα and ß selective inhibitor Gö6976 and siRNA-mediated knockdown of PKCα suppressed Erastin-mediated cell death in RMS. Moreover broad spectrum nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX) inhibitor Diphenyleneiodonium (DPI) and a more selective NOX1/4 isoform inhibitor GKT137831 abrogated Erastin-generated ROS formation, lipid peroxidation and cell death. With this, we demonstrate that RMS are vulnerable to ferroptotic cell death and investigated the molecular mechanism of ferroptosis by unravelling that PKC and NOX could have a pivotal role in ROS-mediated ferroptosis signalling in RMS. In this regard, ferroptosis inducers may act as a possible novel treatment strategy for RMS, especially those with poor clinical outcome.
Onkogene RAS-Mutationen zählen mit einem Vorkommen von ca. 25% zu häufigen Genmutationen in malignen Tumoren. Auch im Rhabdomyosarkom (RMS), dem häufigsten Weichteilsarkom im Kindesalter, findet sich eine hohe Rate an wiederkehrenden RAS-Signalwegmutationen. Dabei scheint ein Zusammenhang zwischen der RMS-Risikostratifizierung und dem Vorkommen von RAS-Mutationen zu bestehen. Da Hochrisiko-RMS im Vergleich zu anderen Tumorentitäten im Kindesalter immer noch mit einer unterdurchschnittlichen Prognose einhergehen, stellen RAS-Mutationen einen interessanten Angriffspunkt für eine zielgerichtete Tumortherapie dar. Hierzu soll diese Arbeit durch eine genauere Charakterisierung der Auswirkungen onkogener RAS-Gene auf das RMS beitragen. Verwendet wurden genetisch modifizierte RMS13 Zellen mit ektoper Expression der onkogenen RAS-Mutationen HRAS12V, KRAS12V oder NRAS12V. Eine bereits gut beschriebene Eigenschaft von RAS ist die Förderung der Zellproliferation. Daneben wurde auch beschrieben, dass RAS Einfluss auf den programmierten Zelltod nehmen und in Abhängigkeit vom zellulären Kontext pro- oder auch antiapoptotisch wirken kann. Daher stellte sich die Frage, welche Auswirkungen onkogene RAS-Mutationen in diesem Kontext auf Rhabdomy-osarkomzellen haben. In dieser Arbeit wird gezeigt, dass die ektope Expression von HRAS12V, KRAS12V oder NRAS12V in RMS13 Zellen zu einer gesteigerten Zellproliferation führt, im Hinblick auf die spontane Zelltodrate jedoch keine Veränderungen bewirkt. Damit stellt die erhöhte Proliferationsrate RAS-mutierter Rhabdomyosarkome einen wichtigen Unterschied zu entsprechenden Tumoren ohne solche Mutationen dar. Chemotherapeutika wie Etoposid und Doxorubicin, die besonders effektiv gegen hochproliferierende Zellen sind, zeigen jedoch keinen signifikanten Unterschied in ihrer Wirksamkeit gegen RMS13 Zellen in Anwesenheit von onkogenem RAS. Damit scheint ein selektives Eingreifen in die proliferationsfördernden Mechanismen nötig zu sein, um RAS-mutierte Zellen gezielt in ihrem Wachstum zu hemmen. Dies verdeutlicht die Notwendigkeit, spezifischer, gezielter Tumortherapien. Neben dem Einfluss auf das Zellwachstum wurden auch Veränderungen in der Redoxhomöostase untersucht. Bisherige indirekte Hinweise auf einen erhöhten oxidativen Stress im RMS in Anwesenheit von RAS-Mutationen können in dieser Arbeit durch den direkten Nachweis erhöhter ROS-Level in RAS-mutierten RMS13 Zellen bestätigt werden. Die akzelerierte ROS-Konzentration lässt vermuten, dass das Überleben von RMS-Zellen mit konstitutiver RAS-Aktivierung in besonderem Maße von antioxidativen Zellstrukturen abhängig sein könnte. Dies könnte sie sensibler gegenüber exogenen Stimuli machen, die zu einer weiteren Erhöhung des oxidativen Stresses führen. Als hervorzuhebendes Ergebnis zeigt diese Arbeit jedoch, dass die ektope Expression von HRAS12V, KRAS12V oder NRAS12V in RMS13 Zellen vor einem oxidativen Zelltod schützt. In Anwesenheit der RAS-Mutationen zeigen RMS13 Zellen einen signifikant geringeren Zellviabilitätsverlust gegenüber einem Eingriff in verschiedene Komponenten des antioxidativen Systems wie durch RSL3 (Glutathion-Peroxidase 4 Inhibitor), Erastin (indirekter Inhibitor der Glutathion-Synthese) oder Auranofin (Thioredoxin-Reduktase-Inhibitor). Dies steht im Gegensatz zu den Erstbeschreibungen, in denen für RSL3 und Erastin eine RAS-selektive Wirkung gezeigt wurde. Als Besonderheit kann der durch RSL3 oder Erastin hervorgerufene Zelltod der RMS13 Zellen als Ferroptose identifiziert werden. Hierbei handelt es sich um eine vor kurzem neu beschriebene Form von programmiertem, oxidativem und eisenabhängigem Zelltod. Diese Arbeit verdeutlicht somit, dass onkogene RAS-Mutationen im RMS gezielt in die Redoxregulation eingreifen, jedoch nur in bestimmten zellulären Kontexten für oxidative Stressoren zu sensibilisieren scheinen. Daneben weist diese Arbeit auch einen protektiven Effekt von onkogenem RAS gegenüber dem dualen PI3K/mTOR-Inhibitor PI-103 in RMS13 Zellen nach. Zusammengenommen deutet dies darauf hin, dass RAS selektiv Einfluss auf durch zytotoxische Stimuli hervorgerufenen Zelltod nimmt. Die Ergebnisse dieser Arbeit, insbesondere der Nachweis einer erhöhten Resistenz gegenüber oxidativen Stressoren in Anwesenheit onkogener RAS-Gene, leisten einen wichtigen Beitrag zur Entwicklung neuer zielgerichteter und selektiver RMS-Therapiestrategien.
Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in many cancers and contribute to apoptosis resistance. Therefore, they represent promising anticancer drug targets. Here, we report that small molecule IAP inhibitors at subtoxic concentrations cooperate with monoclonal antibodies against TRAIL receptor 1 (Mapatumumab) or TRAIL receptor 2 (Lexatumumab) to induce apoptosis in neuroblastoma cells in a highly synergistic manner (combination index <0.1). Importantly, we identify RIP1 as a critical regulator of this synergism. RIP1 is required for the formation of a RIP1/FADD/caspase-8 complex that drives caspase-8 activation, cleavage of Bid into tBid, mitochondrial outer membrane permeabilization, full activation of caspase-3 and caspase-dependent apoptosis. Indeed, knockdown of RIP1 abolishes formation of the RIP1/FADD/caspase-8 complex, subsequent caspase activation and apoptosis upon treatment with IAP inhibitor and TRAIL receptor antibodies. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 inhibits IAP inhibitor- and TRAIL receptor-triggered apoptosis. By comparison, over-expression of the dominant-negative superrepressor IκBα-SR or addition of the TNFα-blocking antibody Enbrel does not inhibit IAP inhibitor- and Lexatumumab-induced apoptosis, pointing to a NF-κB- and TNFα-independent mechanism. Of note, IAP inhibitor also significantly reduces TRAIL receptor-mediated loss of cell viability of primary cultured neuroblastoma cells, underscoring the clinical relevance. By demonstrating that RIP1 plays a key role in the IAP inhibitor-mediated sensitization for Mapatumumab- or Lexatumumab-induced apoptosis, our findings provide strong rationale to develop the combination of IAP inhibitors and TRAIL receptor agonists as a new therapeutic strategy for the treatment of human cancer.
Since Inhibitor of Apoptosis (IAP) proteins are frequently dysregulated in different cancer entities and contribute to apoptosis resistance, pharmacological IAP antagonists are considered to be promising agents for the future development of cancer treatment strategies. IAP antagonists are small-molecule drugs that have been designed to mimic the interaction site of IAP proteins with their endogenous inhibitor Second mitochondrial activator of caspases (SMAC). Thus, they are frequently referred to as SMAC mimetics. Treatment with SMAC mimetics engages an apoptotic program in cancers by affecting different components of the apoptotic machinery. Besides disinhibition of caspases, SMAC mimetics trigger non-canonical nuclear factor-κB (NF-κB) signaling, which induces upregulation of tumor necrosis factor (TNF) α and other NF-κB target genes. In particular, TNFα production has been closely linked to the induction of SMAC mimetic-mediated cell death. The TNFα-dependent para/autocrine loop facilitates the formation of a cytosolic complex consisting of caspase-8, Fas-associated death domain (FADD) and Receptor-interacting protein (RIP) 1, which serves as caspase-8 activation platform and ultimately triggers induction of apoptosis. In the present study, we use the small-molecule bivalent SMAC mimetic BV6 to analyze SMAC-stimulated NF-κB signaling in cancer cell lines of different entities. Interestingly, we identify two novel NF-κB-regulated factors that are both required for SMAC mimetic-induced apoptosis in a context-dependent manner. First, we show that NF-κB-dependent upregulation of death receptor 5 (DR5) can serve as an alternative mechanism of BV6-mediated cell death. We demonstrate that BV6 treatment induces NF-κB-dependent but largely TNFα -independent apoptosis in A172 glioblastoma cells. By using an unbiased whole genome expression analysis approach, we identify DR5 as a critical NF-κB target gene, which substitutes TNFα and is indispensable for BV6-initated cell death in A172 cells. Second, we demonstrate that Interferon regulatory factor (IRF) 1 is required for BV6-induced TNFα production and apoptosis. Our study provides evidence that IRF1 closely cooperates with the NF-κB network in BV6-mediated cell death and additionally alters expression of selective SMAC mimetic-induced target genes. Furthermore, we show that BV6 treatment triggers secretion of a set of proinflammatory cytokines and increases attraction of monocytes to BV6-treated tumor cells in an IRF1-dependent manner. In summary, our work supports the notion that NF-κB-regulated factors are critically required for SMAC mimetic-initiated apoptosis. We show that IRF1 is indispensable for TNFα production and cell death in BV6-sensitive cell lines and that also DR5 can serve as a proapoptotic NF-κB-controlled factor in BV6-induced apoptosis besides TNFα. Furthermore, this study contributes to an improved understanding on non-apoptotic functions of SMAC mimetics, as IRF1 additionally influences expression levels of proinflammatory cytokines and attraction of immune cells. Thus, our work provides novel insights into the regulation of SMAC mimetic-induced signaling events, which is crucial for the translation of SMAC mimetics for use in clinical application.
Protein quality control systems (PQC), i.e. UPS and aggresome-autophagy pathway, have been suggested to be a promising target in cancer therapy. Simultaneous pharmacological inhibition of both pathways have shown increase efficacy in various tumors, such as ovarian and colon carcinoma. Here, we investigate the effect of concomitant inhibition of 26S proteasome by FDA-approved inhibitor Bortezomib, and HDAC6, as key mediator of the aggresome-autophagy system, by the highly specific inhibitor ST80 in rhabdomyosarcoma (RMS) cell lines. We demonstrated that simultaneous inhibition of 26S proteasome and selective aggresome-autophagy pathway significantly increases apoptosis in all tested RMS cell lines. Interestingly, we observed that a subpopulation of RMS cells was able to survive the co-treatment and, upon drug removal, to recover similarly to untreated cells. In this study, we identified co-chaperone BAG3 as the key mediator of this recovery: BAG3 is transcriptionally up-regulated specifically in the ST80/Bortezomib surviving cells and mediates clearance of cytotoxic protein aggregates by selective autophagy. Impairment of the autophagic pathway during the recovery phase, both by conditional knock-down of ATG7 or by inhibition of lysosomal degradation by BafylomicinA1, triggers accumulation of insoluble protein aggregates, loss of cell recovery and cell death similarly to stable short harpin RNA (shRNA) BAG3 knock-down. Our results are the first demonstration that BAG3 mediated selective autophagy is engaged to cope with proteotoxicity induced by simultaneous inhibition of constitutive PQC systems in cancer cell lines during cell recovery. Moreover, our data give new insights in the regulation of constitutive and on demand PQC mechanisms pointing to BAG3 as a promising target in RMS therapy.
Investigating the inhibition of anti-apoptotic BCL-2 family proteins in pediatric cancer cells
(2020)
Cancer is amongst the leading causes of death in childhood. Rhabdomyosarcoma (RMS) is the most frequently occurring soft tissue sarcoma in children and adolescents. It presumably arises from mesenchymal progenitors of skeletal muscle cells and presents with different subtypes that differ both histologically and genetically. Osteosarcoma (OS) and Ewing sarcoma (ES) are the most frequently diagnosed pediatric bone tumors. Even though the prognosis of these cancer entities improved significantly during recent decades, the survival rates are currently stagnating. Especially, dismal prognosis of relapsed and metastasizing cases of these malignancies urgently call for novel treatment options. BCL-2 proteins are vital guardians that control intrinsic apoptosis. Furthermore, it was shown that BCL-2 proteins critically regulate apoptosis in pediatric solid tumors. BH3 mimetics are small molecules that bind and inhibit anti-apoptotic BCL-2 proteins. They have already been investigated as cancer therapeutics for several years and show first encouraging clinical results. Therefore, we hypothesized that targeting BCL-2, MCL-1 and BCL-XL might be a promising approach to treat RMS, OS and ES.
In this study, we aimed to comprehensively evaluate the potential of anti-apoptotic BCL-2 family proteins as therapeutic targets for pediatric solid tumors such as RMS, OS and ES.
Notably, RMS, OS and ES cells largely expressed the most relevant BCL-2 family protein members. However, cells were widely insensitive to single pharmacological inhibition of either BCL-XL, BCL-2 or MCL-1 by A-1331852, ABT-199 and S63845, respectively. This finding was independent of their BCL-2 family protein expression levels. Significantly, co-administration of A-1331852 and S63845 induced cell death in RMS, OS and ES cell lines in a highly synergistic manner. Transient silencing of MCL-1 and/or BCL-XL verified the co-dependency of RMS cells on these proteins for survival. Importantly, A-1331852/S63845 co-treatment was more efficient in causing cell death in RMS, OS and ES cells than either inhibitor combined with ABT-199. Efficacy of A-1331852/S63845 co-treatment could be additionally demonstrated in a primary sample of pediatric malignant epithelioid mesothelioma.
Mechanistically, concomitant A-1331852/S63845 treatment mediated rapid intrinsic apoptosis involving swift loss of the mitochondrial outer membrane potential as well as activation of caspases-3, -8 and -9. An observed caspase dependent loss of MCL-1 might further amplify the A-1331852/S63845 triggered pro-death signaling. Furthermore, we identified BAX and BAK as key mediators of apoptosis caused by dual inhibition of MCL-1 and BCL-XL. A-1331852/S63845 induced cell death was relying on BAX and/or BAK in a cell line dependent manner. Interestingly, treatment with A-1331852 and S63845 liberated BAK from its interaction with MCL-1 and BCL-XL. Moreover, BAX and BAK were activated and interacted with each other to form a pore in the outer mitochondrial membrane. Further, in RD cells BIM and NOXA partially contributed to A-1331852/S63845 mediated cell death. Consistently, in this cell line BIM and NOXA were disrupted from their binding to BCL-XL and MCL-1 by A-1331852 and S63845, respectively. However, BH3 only proteins were not involved in A-1331852/S63845 induced cell death in Kym-1 cells. Therefore, we concluded that BH3 only proteins played only a marginal and cell line dependent role in mediating cell death caused by MCL-1 and BCL-XL co-repression.
Notably, A-1331852/S63845 co-treatment spared non-malignant fibroblasts, myoblasts and peripheral blood mononuclear cells, which suggests a therapeutic window for its application in vivo. Besides, we could demonstrate that sequential BH3 mimetic treatment still significantly induced cell death, albeit to minor extents compared to its dual administration. Importantly, we successfully evaluated concomitant treatment with A-1331852 and S63845 in multicellular RMS spheroids and in an in vivo embryonic chicken model of RMS. These findings stress the high transcriptional relevance of A-1331852/S63845 as an emerging novel cancer regimen.
Collectively, the thesis at hand explored the great potential of co-treatment with A-1331852 and S63845 in pediatric solid tumors and unveiled the underlying molecular mechanisms of cell death in RMS. Together, the current investigations support further preclinical and clinical studies to evaluate the effect of dual MCL-1 and BCL-XL targeting in pediatric solid tumors.
BH3 mimetics are novel anticancer therapeutics that induce apoptosis by targeting anti‐apoptotic BCL‐2 proteins. Highly specific inhibitors of the main anti-apoptotic proteins BCL-2, BCL‐XL and MCL‐1 promise new opportunities for the treatment of AML. However, it is currently unclear which of these anti-apoptotic BCL-2 proteins represents the most promising target in AML. Therefore, we investigated the effect of BH3 mimetics targeting either BCL-2 (ABT-199, S55746), BCL-XL (A-1331852) or MCL-1 (S63845) on eleven AML cell lines. Drug sensitivity screening revealed heterogeneous sensitivity towards the different BH3 mimetics, with the best responses observed upon targeting of MCL-1. Selected cell lines that displayed sensitivity towards the specific BH3 mimetics underwent intrinsic apoptosis, which was characterized by loss of mitochondrial membrane potential, exposure of phosphatidylserine and activation of caspases. Furthermore, S63845 turned out to displace BIMS and NOXA from MCL-1 to induce apoptotic cell death. Importantly, the translational relevance of this study was demonstrated by experiments in primary AML blasts, which displayed similar sensitivity towards BH3 mimetics as the cell lines did. Additionally, experiments with nonmalignant cells could confirm the clinical relevance of the MCL-1 inhibitor. There we could show, that S63845 does not cause cytotoxicity on HPCs at efficacious doses.
In conclusion, our findings reveal that the inhibition of BCL-2 proteins, especially MCL-1, by BH3 mimetics can be a promising strategy in AML treatment.
HCMV ist ein Pathogen mit einer weltweit sehr hohen Prävalenz und stellt nach wie vor ein großes Problem für Immunsupprimierte (Transplantations- und AIDS-Patienten) und für ungeborene Kinder dar. Seine Pathogenese ist weiterhin nur unzureichend bekannt, eine Impfprophylaxe existiert nicht und es stehen nur wenige, unzureichende Medikamente zur Therapie zur Verfügung. Auch ein verstärkender Einfluss des Virus auf die Tumormalignität ist gezeigt. Aber die der viralen Onkomodulation zugrundeliegenden Mechanismen, insbesondere die Frage, wie und wann Tumorwachstum und -invasion durch HCMV beschleunigt werden, bedürfen weiterer Aufklärung.
Die immediate early-Proteine IE1 und IE2 sind wichtige Regulatorproteine einer HCMV-Infektion – insbesondere IE1, welches einen entscheidenden Einfluss auf die Genexpression der Wirtszelle nimmt. Es wurde a-priori die Hypothese erstellt, dass über einen positiven Einfluss von IE1 auf die Enterokinaseexpression die Trypsin-vermittelte Tumorinvasion gefördert wird.
Im ersten Teil dieser Arbeit wurde ein effizientes, zuverlässiges Protokoll zur Herstellung IE1- und IE2-exprimierender Tumorzelllinien mit Hilfe von lentiviralem Gentransfer ausgearbeitet. Damit verbunden konnten zwei persistent-transduzierte humane Tumorzelllinien, U-251 MGIE1 und U-251 MGIE2, mit hohen Expressionsraten etabliert werden. Weiter konnte im zweiten Teil der Arbeit, sowohl im Akutmodell als auch an persistent-transduzierten Zellen, gezeigt werden, dass die Enterokinaseexpression nicht im Zusammenhang mit der IE1-Expression steht.
Durch die Erarbeitung des Transduktions-Protokolls und die Etablierung der persistent-transduzierten humanen Tumorzelllinien ist es nun möglich, die isolierten Auswirkungen von IE1 und IE2 auf humane Tumorzellen zu untersuchen. Die Widerlegung der Hypothese über die Beeinflussung der Enterokinaseexpression durch IE1 liefert eine Erkenntnis, um die Mechanismen der erhöhten Tumorinvasion besser verstehen zu können. Insgesamt konnte durch diese Arbeit ein Beitrag geliefert werden, die durch HCMV verursachten onkomodulatorischen Effekte noch besser nachvollziehen zu können. Dies wiederum kann künftig dazu dienen therapeutische Strategien, welche der Invasion und Metastasierung entgegen wirken, zu verbessern bzw. zu entwickeln.
Das Neuroblastom ist der häufigste extrakranielle solide Tumor des Kindesalters. Bei Diagnosestellung befinden sich die meisten Patienten bereits in fortgeschrittenen Tumorstadien; trotz intensiver multimodaler Therapie überleben nur 30-40% der Hochrisikopatienten die Erkrankung. Zum Therapieversagen führt in den meisten Fällen eine Resistenzentwicklung des Tumors gegenüber den Chemotherapeutika. Die Entdeckung neuer effektiver Therapieansätze und Überwindung der Chemoresistenz durch Resensibilisierung der Tumorzellen ist daher ein dringendes Forschungsanliegen.
Zur Charakterisierung der Zelllinien im ersten Teil dieser Arbeit wurde die Zellmorphologie, die Gen- und Proteinexpression verschiedener Differenzierungs- bzw. Krebsstammzell-Marker und das Anoikis-Verhalten der Neuroblastomzellen UKF-NB-2, UKF-NB-3 und UKF-NB-6 sowie ihrer Cisplatin- und Carboplatin-resistenten Sublinien untersucht. Der zytomorphologischen Phänotyp der untersuchten Zellen ließ keine eindeutigen Schlüsse auf eine neuronale, indifferente oder nicht-neuronale Differenzierung der Zellen zu. Gemessen an der Expression der neuronalen Marker NCAM, TH und der Neurofilamente L, M und H zeigte jedoch die Mehrzahl der untersuchten Cisplatin- und Carboplatin-resistenten Sublinien einen signifikanten Verlust der neuronalen Differenzierung im Vergleich zu ihren parentalen Zellen. Dieser Effekt war auch durch eine temporäre Platinkarenz nicht vollständig reversibel.
Der EGF-Rezeptor, dessen Überexpression als negativer prognostischer Marker für den Therapieerfolg gilt, wurde von allen untersuchten Zelllinien exprimiert, es ließ sich jedoch keine signifikant verstärkte Expression in den resistenten Sublinien nachweisen.
Eine Krebsstammzelle ließ sich in den untersuchten Zelllinien bei schwacher bis fehlender Stammzellmarkerexpression von CD133 und c-Kit nicht eindeutig identifizieren.
Die Resistenz gegenüber Anoikis ist eine Grundvoraussetzung für die Metastasierung von Tumorzellen. Bei den in dieser Arbeit untersuchten Neuroblastomzelllinien zeigten 3 von 8 Zelllinien, UKF-NB-2, UKF-NB-2rCDDP500 und UKF-NB-6, eine Anoikis-Resistenz. UKF-NB-3 sowie ihre beiden Sublinien waren Anoikis-sensibel, sie zeigten alle einen signifikanten Viabilitätsverlust durch Kultivierung auf Poly-HEMA-Beschichtung und daraus resultierendem Adhärenzverlust. Bei UKF-NB-6 nahm durch den Erwerb der Platinresistenz die Toleranz gegenüber Anoikis ab, wie man an dem signifikanten Viabilitätsverlust der Sublinien UKF-NB-6rCDDP1000 und UKF-NB6rCarbo1000 unter nicht-adhärenten Bedingungen sieht. Die Ausbildung der Cisplatin- und insbesondere der Carboplatinresistenz geht hier mit einer signifikant verstärkten Sensitivität der Zellen gegenüber Anoikis einher. Ein synergistischer Effekt auf die Zellviabilität durch Anoikis-induzierende PolyHEMA-Beschichtung und simultane Cisplatin- oder Carboplatin-Exposition ließ sich jedoch nicht beobachten.
Im zweiten Teil dieser Arbeit wurden die durch die Connectivity Map ermittelten potentiellen Resensitizer für Cisplatin (Pararosanilin, Tolbutamid, Fludrocortison, 12,13-EODE und Topiramat) und deren Wirkung auf die Viabilität der Neuroblastomzelllinien (IMR-5, NGP, SK-N-AS, UKF-NB-2, UKF-NB-3 und UKF-NB-6) sowie ihrer Cisplatin-resistenten Sublinien untersucht.
Hierbei zeigte die Kombinationstherapie von Cisplatin mit 12,13-EODE, Topiramat oder Fludrocortison keine signifikante Reduktion der Zellviabilität im Vergleich zur Therapie mit Cisplatin alleine. Ein z. T. signifikanter Anstieg des IC50-Werts von Cisplatin in den getesteten parentalen Zellen und resistenten Sublinien ließ eher einen desensibilisierenden Effekt dieser Stoffe gegenüber Cisplatin vermuten.
Die Kombination von Cisplatin mit Pararosanilin oder Tolbutamid hingegen hatte einen deutlich wachstumshemmenden Effekt auf alle untersuchten resistenten Sublinien. Die IC50-Werte von Cisplatin wurden hier in fast allen Zelllinien signifikant reduziert, z. T. bis um den Faktor 2,45, was einer Halbierung der Cisplatindosis entspricht. Pararosanilin und Tolbutamid erwiesen sich somit als mögliche Resensitizer für Cisplatin in Cisplatin-resistenten Neuroblastomzellen.
Diese Daten lassen erkennen, dass die Connectivity Map ein vielversprechendes Werkzeug in der gezielten Therapie von chemoresistenten Neuroblastomen sein kann. In Kombination mit bisher gängigen Therapieschemata könnten Resensitizer den Erfolg der Behandlung möglicherweise deutlich verbessern. Die mögliche Toxizität der identifizierten Resensitizer, insbesondere Pararosanilin, und damit den tatsächlichen Stellenwert dieses Therapieansatzes wird man jedoch zunächst in vivo noch weiter untersuchen müssen.
Resistance in glucocorticoid-induced apoptosis is associated with poor prognosis for long term survival in childhood acute lymphoblastic leukemia (ALL). As Smac mimetics have been shown to reactivate apoptosis by antagonizing Inhibitor of Apoptosis (IAP) proteins, we investigate the potential of the Smac mimetic BV6 to overcome glucocorticoid-resistance in ALL. This study shows that BV6 synergistically cooperates with glucocorticoids to trigger apoptosis and to suppress clonogenic growth of pediatric ALL cells. Of note, the BV6/glucocorticoid combination treatment also induces cell death in cells having defects in the apoptotic signaling cascade by inducing a switch from apoptotic to necroptotic cell death. The clinical relevance of our novel combination treatment is underscored by parallel experiments in primary pediatric ALL samples, in which glucocorticoids and BV6 act together to induce cell death in a synergistic manner. Importantly, the addition of BV6 enhances the anti-leukemic effects of glucocorticoids in an in vivo mouse model of pediatric ALL without causing substantial side effects, highlighting the potency of a BV6/glucocorticoid combination treatment. In contrast, BV6 does not increase cytotoxicity of glucocorticoids against several non-malignant cell types of the lympho-hematopoietic system. Furthermore, we have identified the novel underlying mechanism of BV6/glucocorticoid-induced apoptosis by showing that BV6 and glucocorticoids synergistically act together to promote assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing cell death complex. Ripoptosome assembly is critically required for BV6/Dexamethasone-induced cell death, since genetic silencing of its members, i.e. RIP1, reduces ROS production, caspase activation and most importantly cell death induction. BV6/glucocorticoid combination treatment promotes ripoptosome assembly by inhibition of both of its negative regulators, IAP proteins and cFLIP. Thus, we identify that BV6 and glucocorticoids cooperate together to reduce cIAP1, cIAP2 and XIAP protein levels and cFLIP expression. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, since blocking antibodies for TNFα, TRAIL or CD95L or genetic silencing of their corresponding receptors fail to rescue BV6/glucocorticoid-induced cell death. In summary, this study shows that the Smac mimetic BV6 sensitizes for glucocorticoid-induced apoptosis by promoting ripoptosome assembly with important implications for the treatment of childhood ALL.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
Cancer cells, in general and especially Rhabdomyosarcoma (RMS) cells have been reported to be highly susceptible to oxidative stress. Based on this knowledge we examined whether the inhibition of the two main antioxidant defense pathways, i.e. the thioredoxin (TRX) and the glutathione (GSH) system, represents a possible new strategy to induce cell death in RMS. To do so, we combined the -glutamylcysteine synthetase (γGCL) inhibitor buthionine sulfoximine (BSO) or the cystine/glutamate antiporter (xc-) inhibitor erastin (ERA), both GSH depleting enzymes, with the thioredoxinreductase (TrxR) inhibitor auranofin (AUR) to evaluate synergistic cell death in the alveolar RMS (ARMS) cell line RH30 and the embryonal RMS (ERMS) cells RD.
Furthermore, we tried to unravel the underlying molecular mechanisms of AUR/BSO or AUR/ERA treatment in RMS cells. Thereby we showed that AUR/BSO as well as AUR/ERA treatment leads to proteasome inhibition characterized by the accumulation of ubiquitinated proteins, which is in agreement with the already published ability of AUR to inhibit proteasomeassociated deubiquitinases (DUBs) aside from TrxR. As a consequence, the protein levels of ubiquitinated short-lived proteins, like NOXA and MCL-1, increase upon treatment with AUR/BSO or AUR/ERA. Consistently, we could detect an increased binding of NOXA to MCL-1. Interestingly, not only NOXA protein levels but also mRNA levels rise upon treatment, pointing to a transcriptional regulation of pro-apoptotic NOXA through AUR/BSO or AUR/ERA combination treatment. The fact that siRNA mediated knockdown of NOXA rescues cells from combination treatment-induced cell death strengthens the role of NOXA as an important regulator of cell death induction. Apart from proteasome inhibition and subsequent NOXA accumulation, AUR cooperates with BSO or ERA to trigger BAX/BAK activation, which is needed for cell death induction, too. Additionally, loss of mitochondrial membrane potential (MMP) as well as caspase activation and PARP cleavage is detected after treatment of RMS cells with AUR/BSO or AUR/ERA.
Except of apoptotic cell death we also detected features of iron-dependent ferroptosis after treatment with AUR/BSO or AUR/ERA. This is not surprising, since BSO and ERA already have been described to induce ferroptotic cell death. Although lipid peroxidation takes place in both cell lines, only in RH30 cells, cell death seems to be partially ferroptosis-dependent, since especially in this cell line AUR/BSO- or AUR/ERA-induced cell death can be rescued with different ferroptosis inhibitors.
Although both combination treatments, AUR/BSO as well as AUR/ERA, induce production of reactive oxygen species (ROS), only the thiol-containing ROS scavengers GSH and its precursor N-acetylcysteine (NAC), but not the non-thiolcontaining antioxidant α-Tocopherol (α-Toc), consistently prevent proteasome inhibition, NOXA accumulation and cell death.
Additionally, we demonstrated that BSO and ERA abolish AUR-mediated upregulation of GSH thereby releasing the AUR cytotoxic effect on RMS cells, in line with the described ability of cysteines to inhibit the function of AUR. Together, this points to the conclusion that GSH depletion, rather than an increase in ROS levels, is important for AUR/BSO- or AUR/ERA-induced cell death.
In conclusion, through revealing that the antitumor activity of AUR is enhanced in combination with GSH depleting agents, we identified redox homeostasis as a new and promising target for the treatment of RMS cells.
Necroptosis is a programmed cell death pathway that is implicated in a variety of human diseases. In recent years, increasing knowledge has been gained on the necroptotic signaling cascade. Nevertheless, the role of reactive oxygen species (ROS) in necroptosis is still ambiguous. In this study, we reveal that ROS critically regulate BV6/TNFα-induced necroptotic signaling in FADD-deficient Jurkat cells and in zVAD-treated MV4-11 cells. We show that several ROS scavengers such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), α-tocopherol (αToc) and ethyl pyruvate (EP) significantly reduce ROS production and BV6/TNFα–induced cell death. Importantly, ROS are produced prior to cell death induction and promote the assembly of the Receptor-interacting protein kinase (RIP)1/RIP3 necrosome complex via a potential positive feedback loop since on the one hand radical scavengers diminish RIP1/RIP3 necrosome formation and since on the other hand RIP1 or RIP3 silencing attenuates ROS production. Furthermore, the deubiquitinase CYLD contributes to BV6/TNFα-induced ROS generation, necrosome assembly and cell death since CYLD knockdown attenuates all these events. Of note, knockdown of the downstream effector protein mixed lineage kinase domain like (MLKL) only partly reduces BV6/TNFα-triggered ROS production and cell death and does not affect necrosome formation. Contrary to expectations, the MLKL inhibitor Necrosulfonamide (NSA) not only decreases BV6/TNFα-stimulated ROS production and cell death but also attenuates RIP1/RIP3 necrosome assembly pointing to additional and MLKL-independent anti-necroptotic effects of NSA. Interestingly, silencing of the potential necroptotic excecutors mitochondrial proteins phosphoglycerate mutase family member 5 (PGAM5) or Dynamin-related protein 1 (Drp1) does not affect BV6/TNFα-induced cell death. Consistently, mitochondrial perturbations are not implicated in BV6/TNFα-induced cell death since mitochondrial membrane potential and respiration remain stable along with to BV6/TNFα-triggered necroptosis induction. Interference with the mitochondrial potential by depolarizing agents such as FCCP reduces BV6/TNFα-induced necroptosis indicating that proper mitochondrial function or a well-defined redox status is required for necroptotic cell death execution. This study demonstrates that ROS are critically involved in BV6/TNFα-induced necroptosis and thus provides novel insights into the redox regulation of necroptotic signaling.
Biologische Signalwege bilden komplexe Netzwerke aus, um die Zellantwort sensibel regulieren zu können. Systembiologische Ansätze werden eingesetzt, um biologische Systeme anhand von Computer-gestützten Modellen zu untersuchen. Ein mathematisches Modell erlaubt, neben der logischen Erfassung der Regulation des biologischen Systems, die systemweite Simulation des dynamischen Verhaltens und Analyse der Robustheit und Anfälligkeit.
Der TNFR1-vermittelte Signalweg reguliert essenzielle Zellvorgänge wie Entzündungsantworten,
Proliferation und Zelltod. TNFR1 wird von dem Zytokin TNF-α stimuliert und fördert daraufhin die Bildung verschiedener makromolekularer Komplexe, welche unterschiedliche Zellantworten einleiten, von der Aktivierung des Transkriptionsfaktors NF-κB, welcher die Expression von proliferationsfördernden Genen reguliert, bis zu zwei Formen des Zelltods, der Apoptose und der Nekroptose. Die Regulation der verschiedenen Zellantworten wird auch als molekularer Schalter bezeichnet. Die exakten molekularen Vorgänge, welche die Zellantwort modulieren, sind noch nicht vollständig entschlüsselt. Eine Fehlregulation des Signalwegs kann chronische Entzündungen hervorrufen oder die Entstehung von Tumoren fördern.
In dieser Thesis haben wir die neuesten Erkenntnisse der Forschung des TNFR1-Signalwegs anhand von umfangreichen Interaktionsdaten aus der Literatur erstmals in einem Petrinetz-Modell erfasst und analysiert. Das manuell kuratierte Modell umfasst die sequenziellen Prozesse der NF-κB-Aktivierung, Apoptose und Nekroptose und berücksichtigt den Einfluss posttranslationaler Modifikationen.
Weiterhin wurden Analysemethoden für Signalwegs-Modelle entwickelt, welche die spezifischen Anforderungen dieser biologischen Systeme berücksichtigen und eine biologisch motivierte Netzwerkanalyse ermöglichen. Die Manatee-Invarianten identifizieren Signalflüsse im Gleichgewichtszustand in Modellen, die Zyklen aufweisen, und werden als Linearkombination von Transitions-Invarianten gebildet. Diese Signalflüsse erfassen idealerweise einen Prozess von der Rezeptorstimulation zur Zellantwort in einem Modell eines Signalwegs. Die Bestimmung aller möglichen Signalflüsse in Modellen von Signalwegen ist eine notwendige Voraussetzung für weitere biologisch motivierte Analysen, wie die in silico-Knockout Analyse. Wir haben ebenfalls ein neues Konzept zur Untersuchung von in silico-Knockouts vorgestellt. Die Effekte der in silico-Knockouts auf einzelne Komplexe und Prozesse des Signalwegs werden in der in silico-Knockout-Matrix repräsentiert. Wir haben die Software-Anwendung isiKnock entwickelt, welche beide Konzepte kombiniert und eine systematische Knockout-Analyse von Petrinetz-Modellen unterstützt.
Das Petrinetz-Modell des TNFR1-Signalwegs wurde auf seine elementaren Eigenschaften geprüft und die etablierten Analysen wie Platz-Invarianten und Transitions-Invarianten durchgeführt. Hierbei konnten die Transitions-Invarianten nicht in allen Fällen komplette biologische Signalflüsse beschreiben. Wir haben ebenfalls die neu vorgestellten Methoden auf das Petrinetz-Modell angewandt. Anhand der Manatee-Invarianten konnten wir die zusammenhängenden Signalflüsse identifizieren und nach ihrem biologischen Ausgang klassifizieren sowie die Auswirkungen der Rückkopplungen untersuchen. Wir konnten zeigen, dass die survival-Antwort durch die Aktivierung von NF-κB am häufigsten auftritt, danach die Apoptose, gefolgt von der Nekroptose. Die alternativen Signalflüsse in Form der Manatee-Invarianten spiegeln die Robustheit des biologischen Systems wider. Wir führten eine ausgiebige in silico-Knockout-Analyse basierend auf den Manatee-Invarianten durch, um die Proteine des Signalwegs nach ihrem Einfluss einzustufen und zu gruppieren. Die Proteine des Komplex I wiesen hierbei den größten Einfluss auf, angeführt von der Rezeptorstimulation und RIP1. Wir betrachteten und diskutierten die Regulation des molekularen Schalters anhand der Knockout-Analyse von selektierten Proteinen und deren Auswirkung auf wichtige Komplexe im Modell. Wir identifizierten die Ubiquitinierung in Komplex I sowie die NF-κB-abhängige Genexpression als die wichtigen Kontrollpunkte des TNFR1-Signalwegs. In Komplex II ist die Regulation der Aktivierung der Caspase-Aktivität entscheidend.
Die umfangreiche Netzwerkanalyse basierend auf Manatee-Invarianten und systematischer in silico-Knockout-Analyse verifizierte das Petrinetz-Modell und erlaubte die Untersuchung der Robustheit und Anfälligkeit des Systems. Die neu entwickelten Methoden ermöglichen eine fundierte, biologisch relevante Untersuchung von in silico-Modellen von Signalwegen. Der systembiologische Ansatz unterstützt die Aufklärung der Regulation und Funktion des verflochtenen Netzwerks des TNFR1-Signalwegs.
Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.
Ubiquitination is regarded as one of the key post-translational modifications in nearly all biological processes, endowed with numerous layers of complexity. Deubiquitinating enzymes (DUBs) dynamically counterbalance ubiquitination events by deconjugating ubiquitin signals from substrates. Dysregulation of the ubiquitin code and its negative regulators drive various pathologies, such as neurological disorders and cancer.
The DUB ubiquitin-specific peptidase 22 (USP22) is well-known for its essential role in the human Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, mediating the removal of monoubiquitination events from Histone 2A and 2B (H2A and -B), thereby regulating gene transcription. In cancer, USP22 was initially described as a part of an 11-gene expression signature profile, predicting tumor metastasis, reoccurrence and death after therapy in a wide range of tumor cells. However, novel roles for USP22 have emerged recently, accrediting USP22 essential roles in regulating tumor development as well as apoptotic cell death signaling.
One of the hallmarks of cancer is the evasion of cell death, especially apoptosis, a form of programmed cell death (PCD). Necroptosis, a regulated form of necrosis, is regarded as an attractive therapeutic strategy to overcome apoptosis-resistance in tumor cells, although a profound understanding of the exact signaling cascade still remains elusive. Nevertheless, several ubiquitination and deubiquitination events are described in fine-tuning necroptotic signaling.
In this study, we describe a novel role for USP22 in regulating necroptotic cell death signaling in human tumor cell lines. USP22 depletion significantly delayed TNFa/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFa-induced nuclear factor-kappa B (NF-KB) signaling or TNFa-mediated extrinsic apoptosis. Intriguingly, re-expression of USP22 wildtype in the USP22 knockout background could re-sensitize HT-29 cells to TBZ-induced necroptosis, whereas re-constitution with the catalytic inactive mutant USP22 Cys185Ser did not rescue susceptibility to TBZ-induced necroptosis, confirming the USP22 DUB-function a pivotal role in regulating necroptotic cell death. USP22 depletion facilitated ubiquitination and unexpectedly also phosphorylation of Receptor-interacting protein kinase 3 (RIPK3) during necroptosis induction, as shown by Tandem Ubiquitin Binding Entities (TUBE) pulldowns and in vivo (de)ubiquitination immunoprecipitations. To substantiate our findings, we performed mass-spectrometric ubiquitin remnant profiling and identified the three novel USP22-regulated RIPK3 ubiquitination sites Lysine (K) 42, K351 and K518 upon TBZ-induced necroptosis. Further assessment of these ubiquitination sites unraveled, that mutation of K518 in RIPK3 reduced necroptosis-associated RIPK3 ubiquitination and additionally affected RIPK3 phosphorylation upon necroptosis induction. At the same time, genetic knock-in of RIPK3 K518R sensitizes tumor cells to TNFa-induced necroptotic cell death and amplified necrosome formation.
In summary we identified USP22 as a new regulator of TBZ-induced necroptosis in various human tumor cell lines and further unraveled the distinctive role of DUBs and (de)ubiquitination events in controlling programmed cell death signaling.
Rhabdomyosarcoma (RMS) is the most frequent pediatric soft-tissue sarcoma comprising two major subtypes – the alveolar and the embryonal rhabdomyosarcoma. The current therapeutic regime is multimodal including surgery, radiation and chemotherapy with cytostatic drugs. Although the prognosis for RMS patients has steadily improved to a 5-year overall survival rate of 70% for ERMS and 50% for ARMS, prognosis for subgroups with primary metastases or relapsed patients is still less than 25%, highlighting the need for development of new therapies for these subgroups. Since cancer cells are addicted to their cancer promoting transcriptional program, remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as compelling anticancer strategy. However, in many cancer types BET inhibition was proved cytostatic but not cytotoxic emphasizing the need for combination protocols.
In this study we identify a novel synergistic interaction of the BET inhibitor JQ1 with p110α-isoform-specific Phosphoinositid-3-Kinase (PI3K) inhibitor BYL719 (Alpelisib) to induce mitochondrial apoptosis and global reallocation of BRD4 to chromatin. At first, we showed that JQ1 single treatment had cytostatic effects at nanomolar concentrations and inhibited MYC and Hedgehog (Hh) signaling in RMS known to promote proliferation of RMS. However, JQ1 single treatment barely induced cell death in RMS cells even at concentrations of up to 20 µM (< 20% cell death). Thus, we next tested combination approaches to elicit cell death. Since we previously identified synergistic cell death induction of Hh inhibition and PI3K inhibition in RMS cells we tested JQ1 in combination with the pan-PI3K/mTOR inhibitor PI-103 and the p110α-isoform-specific PI3K inhibitor BYL719. In addition, we tested JQ1 in combination with distinct HDAC inhibitors namely JNJ-26481585, SAHA (Vorinostat), MS-275 (Entinostat) and LBH-589 (Panobinostat) since the synergistic interaction of BET and HDAC inhibition has previously been described for other tumor entities.
Interestingly the synergism of cell death induction of JQ1/BYL719 co-treatment is superior to the synergism of JQ1 with pan-PI3K/mTOR inhibitor PI-103 or the tested HDAC inhibitors as confirmed by calculation of combination index. To investigate the molecular mechanisms underlying the synergy of JQ1/BYL719 co-treatment, we performed RNA-Seq and BRD4 ChIP-Seq experiments. RNA-Seq exhibited, that JQ1/BYL719 co-treatment shifted the overall balance of BCL-2 family gene expression towards apoptosis and increased gene expression of proapoptotic BMF, BCL2L11 (BIM) and PMAIP1 (NOXA) while decreasing gene expression of antiapoptotic BCL2L1 (BCL xL). These changes were verified by qRT-PCR and Western blot. Notably, BRD4 is phosphorylated upon JQ1/BYL719 co-treatment and globally reallocates BRD4 to chromatin. This BRD4 reallocation includes enrichment of BRD4 at the super-enhancer site of BMF, at the super-enhancer, typical enhancer and promoter regions of BCL2L11 (BIM) and at the PMAIP1 (NOXA) promoter, while JQ1 alone, as expected, reduces global chromatin binding of BRD4. Integration of RNA-Seq and BRD4 ChIP-Seq data underlines the transcriptional relevance of reallocated BRD4 upon JQ1/BYL719 co-treatment. Immunopreciptation studies showed, that RMS cells are initially primed to undergo mitochondrial apoptosis since BIM is constitutively bound to antiapoptotic BCL-2, BCL xL and MCL-1. JQ1/BYL719 co-treatment increased BIM expression and its neutralization of antiapoptotic BCL-2, BCL-xL and MCL-1 thereby rebalancing the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis. This promotes activation of BAK and BAX resulting in caspase-dependent apoptosis. The functional relevance of proapoptotic re-balancing for the execution of JQ1/BYL719-mediated apoptosis was confirmed by individual silencing of BMF, BIM, NOXA or overexpression of BCL-2 or MCL-1, which all significantly rescued JQ1/BYL719-induced cell death. Execution of cell death by mitochondrial caspase-dependent apoptosis was veryfied by individual knockdown of BAK and BAX or caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk), which all significantly rescued JQ1/BYL719-induced cell death.
In summary, combined BET and PI3Kα inhibition cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins accompanied by reallocation of BRD4 to transcriptional regulatory elements of BH3-only proteins.
Glioblastoma multiforme accounts for more than 80% of all malignant gliomas in adults and a minor fraction of new annual cases occurs in children. In the last decades, research shed light onto the molecular patterns underlying human malignancies which resulted in a better understanding of the disease and finally an improved long term survival for cancer patients. However, malignancies of the central nervous system and especially glioblastomas are still related to poor outcomes with median survivals of less than 6 months despite extensive surgery, chemotherapy and radiation. Hence, a better understanding of the molecular mechanism driving and sustaining cancerous mutations in glioblastomas is crucial for the development of targeted therapies. Apoptosis, a form of programmed cell death, is an important feature of eukaryotic cells and crucial for the maintenance of multicellular homeostasis. Because apoptosis is a highly complex and tightly regulated signaling pathway, resisting apoptotic stimuli and avoiding cell death is a hallmark of the cancerous transformation of cells. Hence, targeting molecular structures to reestablish apoptotic signaling in tumor cells is a promising approach for the treatment of malignancies. Smac mimetics are a group of small molecular protein inhibitors that structurally derive from an intracellular protein termed Smac and selectively block Inhibitor of apoptosis (IAP) proteins, which are often aberrantly expressed in cancer. Several studies confirmed the antitumoral effects of Smac mimetics in different human malignancies, including glioblastoma, and give rationales for the development of potent Smac mimetics and Smac mimetic-based combination protocols. This study investigates the antitumoral activity of the bivalent Smac mimetic BV6 in combination with Interferon α. Latter is a well characterized cytokine with an essential role in immunity, cell differentiation and apoptosis. This study further aims to address the molecular mechanisms underlying the antitumoral activity of the combination treatment by using well established molecular cell death assays, flow cytometry, western blot analysis, genetic approaches and selective pharmacological inhibition. Since different Smac mimetics and Smac mimetic-based combination therapies are currently under clinical evaluations, findings of this study may have broad implications for the application of Smac mimetics as clinical cancer therapeutics.